WO2024066026A1 - RÉCEPTEUR ANTIGÉNIQUE CHIMÉRIQUE OPTIMISÉ CIBLANT IL13Rα2 ET SON UTILISATION - Google Patents

RÉCEPTEUR ANTIGÉNIQUE CHIMÉRIQUE OPTIMISÉ CIBLANT IL13Rα2 ET SON UTILISATION Download PDF

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WO2024066026A1
WO2024066026A1 PCT/CN2022/136205 CN2022136205W WO2024066026A1 WO 2024066026 A1 WO2024066026 A1 WO 2024066026A1 CN 2022136205 W CN2022136205 W CN 2022136205W WO 2024066026 A1 WO2024066026 A1 WO 2024066026A1
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amino acid
seq
sequence
variant
cells
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钟晓松
白玥
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卡瑞济(北京)生命科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to the field of genetic engineering technology. Specifically, the present invention relates to an optimized chimeric antigen receptor targeting IL13R ⁇ 2, an immune effector cell (e.g., T cell, NK cell) engineered to express the optimized chimeric antigen receptor of the present invention, and the use of the engineered immune effector cell for treating diseases associated with the expression of IL13R ⁇ 2.
  • an immune effector cell e.g., T cell, NK cell
  • Glioblastoma is the most malignant primary brain tumor in adults. Current standard treatment includes surgical resection, radiotherapy and chemotherapy (eg, using temozolomide), however, the five-year overall survival rate is less than 10%.
  • Immunotherapy is a very attractive treatment method that can improve the prognosis of GBM patients without the cytotoxic reactions caused by chemotherapy or radiotherapy.
  • Adoptive immunotherapy using antibodies or T cells is currently the most effective immunotherapy in clinical and experimental settings.
  • immunotherapy based on dendritic cell vaccines has shown encouraging results in the clinical treatment of relapsed and newly diagnosed GBM patients, stabilizing the disease and increasing the survival of patients, these clinical results need to be confirmed by further randomized clinical trials, and published research articles have also shown that it is difficult to induce glioma-specific T cells in patients. Therefore, there is an urgent need to study and develop new immunotherapy methods.
  • Chimeric antigen receptor T cell (CAR-T) immunotherapy uses transgenic technology to express the single-chain variable region (scFv) of monoclonal antibodies that can specifically recognize target antigens on the surface of T cells, while activating the intracellular activation and proliferation signaling domains of T cells (CD3 ⁇ chain and co-stimulatory molecules CD28/4-1BB), thereby enabling T cells to produce highly efficient and specific anti-tumor responses.
  • scFv single-chain variable region
  • CAR-T is currently the most popular cell immunotherapy.
  • Five drugs have been successfully launched in the United States (Novartis' Kymriah, Kite's Yescarta and Tecartus, BMS' Breyanzi and Abecma). Its earliest subject, Emily Whitedhead (an acute lymphoblastic leukemia patient), has been tumor-free for 8 years, which has rekindled the hope of life for countless cancer patients.
  • the high-affinity interleukin-13 receptor ⁇ 2 (IL13R ⁇ 2) is selectively expressed at high frequency by glioblastoma (GBM) as well as several other tumor types.
  • GBM glioblastoma
  • One approach to targeting this tumor-specific receptor is to utilize the corresponding ligand IL-13 conjugated to a cytotoxic molecule.
  • this approach lacks specificity because the lower affinity receptor IL13R ⁇ 1 that binds IL-13 is widely expressed by normal tissues.
  • the object of the present invention is to provide an optimized third-generation chimeric antigen receptor that specifically targets IL13R ⁇ 2 and immune effector cells (e.g., T cells, NK cells) engineered to express the chimeric antigen receptor, for use in treating diseases associated with the expression of IL13R ⁇ 2, such as glioblastoma (GBM), so as to solve the problems existing in the above-mentioned prior art.
  • IL13R ⁇ 2 e.g., T cells, NK cells
  • GBM glioblastoma
  • the present invention provides an optimized chimeric antigen receptor (CAR) polypeptide targeting IL13R ⁇ 2, comprising:
  • An optimized anti-IL13R ⁇ 2 scFv sequence wherein the scFv sequence specifically binds to IL13R ⁇ 2 and comprises:
  • a heavy chain complementary determining region CDR H1 represented by the amino acid sequence KYGVH (SEQ ID NO: 15), or a variant of said CDR H1 having no more than 2 amino acid changes or no more than 1 amino acid change;
  • CDR H2 represented by the amino acid sequence VKWAGGSTDTDSALMS (SEQ ID NO:16), or a variant of said CDR H2 having no more than 2 amino acid changes or no more than 1 amino acid change;
  • CDR L light chain complementary determining region 1 represented by the amino acid sequence TASLSVSSTYLH (SEQ ID NO:18), or a variant of said CDR L1 having no more than 2 amino acid changes or no more than 1 amino acid change;
  • CDR L3 represented by the amino acid sequence HQYHRSPLT (SEQ ID NO: 20), or a variant of said CDR L3 having no more than 2 amino acid changes or no more than 1 amino acid change;
  • amino acid change is an addition, deletion or substitution of an amino acid
  • CD8 hinge region (SEQ ID NO 8), or a CD8 hinge region having at least 80% sequence identity thereto.
  • TM transmembrane region
  • a CD28 transmembrane domain or a variant thereof having 1-5 amino acid modifications for example, the sequence shown in SEQ ID NO:9 or a variant thereof having 1-2 amino acid modifications;
  • a CD4 transmembrane domain or a variant thereof having 1-5 amino acid modifications for example, the sequence shown in SEQ ID NO: 10 or a variant thereof having 1-2 amino acid modifications;
  • a CD8 transmembrane domain or a variant thereof having 1-5 amino acid modifications for example, the sequence shown in SEQ ID NO: 11 or a variant thereof having 1-2 amino acid modifications;
  • a CD28 co-stimulatory domain or a variant thereof having 1-5 amino acid modifications for example, the sequence shown in SEQ ID NO:12 or a variant thereof having 1-2 amino acid modifications;
  • a 4-1BB co-stimulatory domain or a variant thereof having 1-5 amino acid modifications for example, the sequence shown in SEQ ID NO: 13 or a variant thereof having 1-2 amino acid modifications;
  • Stimulatory signal domain which is the CD3 ⁇ signal transduction domain or a variant thereof having 1-10 amino acid modifications, for example, the sequence shown in SEQ ID NO:14 or a variant thereof having 1-10 or 1-5 amino acid modifications.
  • the optimized chimeric antigen receptor (CAR) polypeptide targeting IL13R ⁇ 2 of the present invention comprises:
  • An optimized anti-IL13R ⁇ 2 scFv sequence wherein the scFv sequence specifically binds to IL13R ⁇ 2 and comprises:
  • a heavy chain variable region comprising the sequence of SEQ ID NO:2, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and
  • a light chain variable region comprising the sequence of SEQ ID NO:4, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;
  • an IgG4 hinge region (SEQ ID NO 7), or an IgG4 hinge region having at least 90% or at least 95% sequence identity thereto;
  • CD8 hinge region (SEQ ID NO 8), or a CD8 hinge region having at least 90% or at least 95% sequence identity thereto.
  • TM transmembrane region
  • a stimulatory signaling domain which is the CD3 ⁇ signaling domain shown in SEQ ID NO: 14 or a variant thereof having one amino acid modification;
  • the amino acid modification is the addition, deletion or substitution of amino acids.
  • the optimized chimeric antigen receptor (CAR) polypeptide targeting IL13R ⁇ 2 of the present invention comprises:
  • An optimized anti-IL13R ⁇ 2 scFv sequence wherein the scFv sequence specifically binds to IL13R ⁇ 2 and comprises:
  • TM transmembrane region
  • the optimized chimeric antigen receptor (CAR) polypeptide targeting IL13R ⁇ 2 of the present invention further comprises a signal peptide sequence at the N-terminus, for example, the signal peptide sequence shown in SEQ ID NO: 21,
  • the optimized chimeric antigen receptor (CAR) polypeptide targeting IL13R ⁇ 2 of the present invention has the amino acid sequence shown in SEQ ID NO:22 or an amino acid sequence that has at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identity thereto.
  • the present invention provides a nucleic acid encoding a chimeric antigen receptor (CAR polypeptide) as described herein, a vector comprising a nucleic acid encoding a CAR polypeptide as described herein, and a cell comprising a CAR nucleic acid molecule or vector as described herein, or a cell expressing a CAR polypeptide as described herein, preferably, the cell is an autologous T cell or an allogeneic T cell.
  • CAR polypeptide chimeric antigen receptor
  • the present invention provides a method for producing cells, such as immune effector cells, comprising introducing (e.g., transducing) a nucleic acid molecule (e.g., an RNA molecule, such as an mRNA molecule) encoding a CAR polypeptide described herein, or a vector comprising a nucleic acid molecule encoding a CAR polypeptide described herein into an immune effector cell.
  • a nucleic acid molecule e.g., an RNA molecule, such as an mRNA molecule
  • a vector comprising a nucleic acid molecule encoding a CAR polypeptide described herein into an immune effector cell.
  • the immune effector cells are T cells, NK cells, for example, the T cells are autologous T cells or allogeneic T cells, for example, the immune effector cells are prepared by isolating T cells, NK cells from human PBMCs.
  • a nucleic acid molecule encoding a CAR polypeptide described herein is introduced into primary T cells using a retrovirus to obtain a CAR-T cell of the present invention.
  • the CAR-T cells of the present invention have significant killing activity against glioma U373 cells that highly express IL13R ⁇ 2 and glioma U251 cells that lowly express IL13R ⁇ 2 in vitro, and produce cytokines such as IFN- ⁇ and TNF- ⁇ .
  • the CAR-T cells of the present invention also have the function of killing tumor cells in vivo.
  • the CAR-T cells of the present invention have improved anti-tumor activity.
  • the present invention provides the use of the immune effector cells expressing the CAR polypeptide of the present invention for preparing a medicament for preventing or treating a tumor (e.g., cancer) or providing anti-tumor immunity in a subject, preferably, the tumor is a glioma, more preferably, the tumor is a glioblastoma.
  • a tumor e.g., cancer
  • the tumor is a glioma
  • the tumor is a glioblastoma.
  • the present invention provides the use of the immune effector cells expressing the CAR polypeptide of the present invention for treating a disease associated with the expression of IL13R ⁇ 2 in a subject, comprising administering a therapeutically effective amount of immune effector cells expressing the CAR polypeptide to the subject, wherein the disease associated with the expression of IL13R ⁇ 2 is, for example, glioma, preferably glioblastoma.
  • the present invention provides a method for treating a mammal suffering from a disease associated with the expression of IL13R ⁇ 2, comprising administering to the mammal an effective amount of an immune effector cell expressing a CAR polypeptide of the present invention, for example, wherein the disease associated with the expression of IL13R ⁇ 2 is glioma, preferably, glioblastoma.
  • the present invention uses the anti-IL13R ⁇ 2 scFv sequence for the first time to construct the third-generation CAR-T cells, which proves that it can treat GBM by specifically targeting IL13R ⁇ 2, thereby improving the safety and effectiveness of CAR-T cell treatment of GBM, avoiding rejection reactions or off-target phenomena, and being more conducive to the promotion and clinical application of CAR-T.
  • FIG1 is a schematic diagram of the optimized CAR structure constructed by the present invention, in which SD represents a splice donor, SA represents a splice acceptor, and LTR represents a long terminal repeat.
  • the extracellular domain comprises an optimized scFv sequence against IL13R ⁇ 2 and a short IgG4 hinge region.
  • Figure 2 shows the results of CD107a staining and detection of CD107a expression rate by flow cytometry when the optimized CAR-T cells targeting IL13R ⁇ 2 and containing the anti-IL13R ⁇ 2 scFv sequence were cultured alone.
  • Figure 3 shows the results of CD107a staining of CAR-T cells when the optimized CAR-T cells targeting IL13R ⁇ 2 and containing the anti-IL13R ⁇ 2 scFv sequence were co-cultured with the U373 cell line that highly expresses IL13R ⁇ 2, and the expression rate of CD107a was detected by flow cytometry.
  • Figure 4 shows the results of CD107a staining of CAR-T cells containing anti-IL13R ⁇ 2 scFv sequence targeting IL13R ⁇ 2 when they were co-cultured with U251 cell line with low expression of IL13R ⁇ 2, and the expression rate of CD107a was detected by flow cytometry.
  • Figure 5 shows the analysis results of the difference in CD107a expression rate after co-culture of optimized CAR-T cells targeting IL13R ⁇ 2 containing anti-IL13R ⁇ 2 scFv sequence with U373 cell line or U251 cell line.
  • Figure 6 shows the experimental results of the optimized CAR-T cells targeting IL13R ⁇ 2 and containing the anti-IL13R ⁇ 2 scFv sequence killing the target cells U373 cell line or U251 cell line.
  • Figure 7 shows the results of differential analysis of the killing of target cells U373 cell line or U251 cell line by optimized CAR-T cells targeting IL13R ⁇ 2 and containing anti-IL13R ⁇ 2 scFv sequences.
  • Figure 8 shows the content of cytokine IFN- ⁇ in the supernatant after co-culture of optimized CAR-T cells targeting IL13R ⁇ 2 containing anti-IL13R ⁇ 2 scFv sequence (abbreviated as "IL13R ⁇ 2-CAR-T” in the figure) with IL13R ⁇ 2-positive human GBM cell lines (U373, U251, U87).
  • Figure 9 shows the content of cytokine TNF- ⁇ in the supernatant after co-culture of optimized CAR-T cells targeting IL13R ⁇ 2 containing anti-IL13R ⁇ 2 scFv sequence (abbreviated as "IL13R ⁇ 2-CAR-T” in the figure) with IL13R ⁇ 2-positive human GBM cell lines (U373, U251, U87).
  • Figure 10 shows the RTCA detection results after co-culture of optimized CAR-T cells targeting IL13R ⁇ 2 containing anti-IL13R ⁇ 2 scFv sequence with U373 cell line.
  • FIG. 11 shows the RTCA detection results after co-culture of CAR-T cells targeting IL13R ⁇ 2 and containing an optimized scFv sequence against IL13R ⁇ 2 with the U251 cell line.
  • Figure 12 is the in vivo imaging results of the treatment of the U251 orthotopic animal model with optimized CAR-T cells containing anti-IL13R ⁇ 2 scFv sequences targeting IL13R ⁇ 2 (abbreviated as "IL13R ⁇ 2-CAR-T” in the figure) or CAR-T cells containing IL13 shown in SEQ ID NO: 24 targeting IL13R ⁇ 2 (abbreviated as "IL13-CAR-T” in the figure), wherein 2 ⁇ 10 5 U251 cells were injected into the right brain striatum of female NOD-SCID mice on day 1 (also abbreviated as D1). 3 ⁇ 10 7 CAR-T cells were injected through the tail vein on day 6 (D6).
  • IL13R ⁇ 2-CAR-T CAR-T cells containing IL13 shown in SEQ ID NO: 24 targeting IL13R ⁇ 2
  • IL13-CAR-T 2 ⁇ 10 5 U251 cells were injected into the right brain striatum of female NOD-SC
  • FIG. 13 shows the survival period of the U251 orthotopic animal model treated with optimized CAR-T cells containing anti-IL13R ⁇ 2 scFv sequence targeting IL13R ⁇ 2 (abbreviated as "IL13R ⁇ 2-CAR-T” in the figure) or CAR-T cells containing IL13 shown in SEQ ID NO:24 targeting IL13R ⁇ 2 (abbreviated as "IL13-CAR-T” in the figure).
  • the experiment is the same as FIG. 12.
  • Figure 14 is the in vivo imaging results of the treatment of U373 orthotopic animal model with optimized CAR-T cells containing anti-IL13R ⁇ 2 scFv sequences targeting IL13R ⁇ 2 (abbreviated as "IL13R ⁇ 2-CAR-T” in the figure) or CAR-T cells containing IL13 shown in SEQ ID NO: 24 targeting IL13R ⁇ 2 (abbreviated as "IL13-CAR-T” in the figure), wherein 2 ⁇ 10 5 U373 cells were injected into the right striatum of female NOD-SCID mice on day 1 (also abbreviated as D1). 3 ⁇ 10 7 CAR-T cells were injected through the tail vein on day 6 (D6).
  • IL13R ⁇ 2-CAR-T CAR-T cells containing IL13 shown in SEQ ID NO: 24 targeting IL13R ⁇ 2
  • IL13-CAR-T 2 ⁇ 10 5 U373 cells were injected into the right striatum of female NOD-SCID mice on
  • FIG. 15 shows the survival period of the U373 orthotopic animal model treated with optimized CAR-T cells containing anti-IL13R ⁇ 2 scFv sequence targeting IL13R ⁇ 2 (abbreviated as "IL13R ⁇ 2-CAR-T” in the figure) or CAR-T cells containing IL13 shown in SEQ ID NO:24 targeting IL13R ⁇ 2 (abbreviated as "IL13-CAR-T” in the figure).
  • the experiment is the same as FIG. 14.
  • chimeric receptor refers to a recombinant polypeptide comprising at least an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain.
  • anti-IL13R ⁇ 2 antibody As used herein, the terms “anti-IL13R ⁇ 2 antibody”, “antibody against IL13R ⁇ 2”, “antibody that specifically binds to IL13R ⁇ 2”, “antibody that specifically targets IL13R ⁇ 2”, “antibody that specifically recognizes IL13R ⁇ 2” are used interchangeably and refer to antibodies that can specifically bind to IL13R ⁇ 2. In particular, in some specific embodiments, it refers to an antibody that specifically binds to human IL13R ⁇ 2, in particular, an antibody that specifically binds to human IL13R ⁇ 2 but does not specifically bind to human IL13R ⁇ 1.
  • scFv refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are optionally connected continuously by means of a flexible short polypeptide linker and can be expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the complete antibody from which it is derived.
  • scFv can have a VL variable region and a VH variable region in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and the scFv can comprise VL-linker-VH or can comprise VH-linker-VL.
  • Each heavy chain variable region (VH) and light chain variable region (VL) are respectively composed of four conserved framework regions (FR) and three complementary determining regions (CDR), arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the scFv sequence for IL13R ⁇ 2 contained in the CAR of the present invention is an optimized sequence, for example, a sequence in which the framework region is optimized.
  • the amino acid sequence of the framework region is optimized, thereby retaining the improved binding affinity of the optimized scFv sequence for IL13R ⁇ 2 to IL13R ⁇ 2.
  • CDR region or “CDR” or “hypervariable region” is a region of an antibody variable domain that is highly variable in sequence and forms structurally defined loops ("hypervariable loops") and/or contains antigen contact residues ("antigen contact points").
  • the CDRs are primarily responsible for binding to antigen epitopes.
  • the CDRs of the heavy and light chains are usually referred to as CDR1, CDR2 and CDR3, numbered sequentially starting from the N-terminus.
  • the CDRs located within the antibody heavy chain variable domain are referred to as CDR H1, CDR H2 and CDR H3, while the CDRs located within the antibody light chain variable domain are referred to as CDR L1, CDR L2 and CDR L3.
  • each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example, Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al.
  • CDR can also be determined based on having the same Kabat numbering position as a reference CDR sequence (e.g., any of the CDRs exemplified in the present invention).
  • a reference CDR sequence e.g., any of the CDRs exemplified in the present invention.
  • the present invention when referring to antibody variable regions and specific CDR sequences (including heavy chain variable region residues), it refers to the numbering position according to the Kabat numbering system.
  • CDR is different from antibody to antibody, only a limited number of amino acid positions in CDR are directly involved in antigen binding.
  • the minimum overlapping region can be determined, thereby providing a "minimum binding unit" for antigen binding.
  • the minimum binding unit can be a sub-portion of the CDR.
  • the residues of the rest of the CDR sequence can be determined by the structure and protein folding of the antibody. Therefore, the present invention also contemplates variants of any CDR given herein.
  • the amino acid residues of the minimum binding unit can remain unchanged, while the remaining CDR residues defined according to Kabat or Chothia or AbM can be replaced by conservative amino acid residues.
  • variable region refers to the domain of an antibody heavy chain or light chain that is involved in binding of the antibody to an antigen.
  • the variable domains of the heavy and light chains of natural antibodies generally have similar structures, wherein each domain comprises four conserved framework regions (FRs) and three complementary determining regions (CDRs).
  • FRs conserved framework regions
  • CDRs complementary determining regions
  • binding means that the binding effect is selective for the antigen and can be distinguished from unwanted or non-specific interactions.
  • the ability of an antibody to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA), SPR or biofilm interferometry or other conventional binding assays known in the art.
  • the term "stimulatory molecule” refers to a molecule expressed by a T cell that provides a primary cytoplasmic signaling sequence that regulates the primary activation of the TCR complex in a stimulatory manner in at least some aspect of the T cell signaling pathway.
  • the primary signal is initiated, for example, by the binding of the TCR/CD3 complex to the MHC molecule loaded with a peptide and leads to mediating a T cell response, including but not limited to proliferation, activation, differentiation, etc.
  • the intracellular signaling domain in any one or more CARs of the present invention comprises an intracellular signaling sequence, for example, a primary signaling sequence of CD3 ⁇ .
  • CD3 ⁇ is defined as a protein provided by GenBank Accession No. BAG36664.1 or its equivalent
  • CD3 ⁇ stimulatory signaling domain is defined as amino acid residues from the cytoplasmic domain of the CD3 ⁇ chain that are sufficient to functionally propagate the initial signal necessary for T cell activation.
  • the cytoplasmic domain of CD3 ⁇ comprises residues 52 to residue 164 of GenBank Accession No. BAG36664.1 or equivalent residues from non-human species (e.g., mice, rodents, monkeys, apes, etc.) that are functional homologs thereof.
  • the "CD3 ⁇ stimulatory signaling domain” is the sequence provided in SEQ ID NO:14 or a variant thereof.
  • costimulatory molecule refers to a corresponding binding partner on a cell that specifically binds to a costimulatory ligand to mediate a costimulatory response of the cell (e.g., but not limited to, proliferation).
  • Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an effective immune response.
  • Costimulatory molecules include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, OX40, CD40, GITR, 4-1BB (i.e., CD137), CD27, and CD28.
  • costimulatory molecules are CD28, 4-1BB (i.e., CD137).
  • the costimulatory signaling domain refers to the intracellular portion of a costimulatory molecule.
  • the nucleic acid sequence encoding the CAR of the present invention can be introduced into cells by "transfection”, “conversion” or “transduction”.
  • transfection refers to the introduction of one or more exogenous polynucleotides into host cells by physical or chemical methods.
  • Many transfection techniques are known in the art, and include, for example, calcium phosphate DNA coprecipitation (see, for example, Murray E.J. (ed.), Methods in Molecular Biology, Vol.
  • signaling pathway refers to the biochemical relationships between multiple signaling molecules that play a role in propagating a signal from one part of a cell to another part of the cell.
  • cytokine is a generic term for proteins released by one cell population that act as intercellular mediators on another cell.
  • cytokines include lymphokines, monokines, interleukins (ILs), such as IL-1, IL-1 ⁇ , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-15; tumor necrosis factors, such as TNF- ⁇ or TNF- ⁇ ; and other polypeptide factors, including gamma-interferon.
  • ILs interleukins
  • activation or “activation” of immune cells refers to the ability of immune cells to respond and exhibit the immune function of the corresponding cells known to those skilled in the art at a measurable level. Methods for measuring immune cell activity are also known to those skilled in the art.
  • amino acid change and “amino acid modification” are used interchangeably and refer to the addition, deletion, substitution and other modifications of amino acids. Any combination of the addition, deletion, substitution and other modifications of amino acids can be performed, provided that the final polypeptide sequence has the desired characteristics.
  • the substitution of amino acids is a non-conservative amino acid substitution, i.e., replacing one amino acid with another amino acid having different structures and/or chemical properties.
  • Amino acid substitutions include substitutions with non-naturally occurring amino acids or naturally occurring amino acid derivatives of twenty standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine).
  • Amino acid changes can be produced using genetic or chemical methods known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, etc. Methods for changing amino acid side chain groups by methods other than genetic engineering (such as chemical modification) may be useful.
  • conservative sequence modification and “conservative sequence change” refer to amino acid modifications or changes that do not significantly affect or change the characteristics of the CAR or its constituent elements containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the CAR of the present invention or its constituent elements by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative substitutions are amino acid substitutions in which amino acid residues are replaced by amino acid residues with similar side chains. Families of amino acid residues with similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • non-polar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • ⁇ -side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine.
  • one or more amino acid residues within the CAR of the present invention can be replaced with other amino acid residues from the same side chain family, and the function of the altered CAR can be tested using the functional assays described here
  • autologous refers to any substance that is derived from the same individual into which the substance is later reintroduced.
  • allogeneic refers to any material that is derived from a different animal of the same species as the individual into which the material is introduced. Two or more individuals are said to be allogeneic to each other when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species can be genetically dissimilar enough to interact antigenically.
  • xenogeneic refers to a graft derived from an animal of a different species.
  • apheresis refers to an art-recognized in vitro method by which a donor's or patient's blood is removed from the donor or patient and passed through a device that separates selected specific components and returns the remainder to the donor's or patient's circulation, e.g., by retransfusion.
  • apheresis sample a sample obtained using apheresis is referred to.
  • immune effector cell refers to a cell that participates in an immune response, e.g., participates in promoting an immune effector reaction.
  • immune effector cells include T cells, e.g., ⁇ / ⁇ T cells and ⁇ / ⁇ T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid derived phagocytes.
  • Immuno effector function refers to, for example, a function or response of an immune effector cell that enhances or promotes immune attack on a target cell.
  • an immune effector function or response refers to a T cell or NK cell property that promotes killing of target cells or inhibits growth or proliferation of target cells.
  • primary stimulation and co-stimulation are examples of immune effector functions or responses.
  • effector function refers to a specialized function of a cell.
  • the effector function of a T cell may be, for example, cytolytic activity or helper activity, including the secretion of cytokines.
  • T cell activation or “T cell activation” are used interchangeably and refer to one or more cellular responses of T lymphocytes, particularly cytotoxic T lymphocytes, selected from the group consisting of proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers.
  • the chimeric antigen receptors of the present invention are capable of inducing T cell activation. Suitable assays for measuring T cell activation are described in the Examples and are known in the art.
  • lentivirus refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in their ability to infect non-dividing cells; they can deliver significant amounts of genetic information to host cells, making them one of the most efficient methods of gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses.
  • lentiviral vector refers to a vector derived from at least a portion of a lentiviral genome, and particularly includes self-inactivating lentiviral vectors as provided in Milone et al., Mol. Ther. 17(8): 1453–1464 (2009).
  • Other examples of lentiviral vectors that can be used clinically include, but are not limited to, lentiviral vectors from Oxford BioMedica. Gene delivery technology, LENTIMAX TM vector system from Lentigen, etc.
  • Non-clinical types of lentiviral vectors are also available and known to those skilled in the art.
  • disease associated with expression of IL13R ⁇ 2 refers to any condition caused by, exacerbated by, or otherwise associated with increased expression or activity of IL13R ⁇ 2.
  • mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).
  • domesticated animals e.g., cattle, sheep, cats, dogs, and horses
  • primates e.g., humans and non-human primates such as monkeys
  • rabbits e.g., mice and rats
  • rodents e.g., mice and rats.
  • the individual or subject is a human.
  • tumor and cancer are used interchangeably herein and encompass both solid and liquid tumors.
  • Tumor immune escape refers to the process by which a tumor evades immune recognition and clearance.
  • tumor immunity is “treated” when such escape is weakened and the tumor is recognized and attacked by the immune system.
  • tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.
  • treatment refers to slowing down, interrupting, blocking, alleviating, stopping, reducing, or reversing the progress or severity of existing symptoms, illnesses, conditions, or diseases.
  • the desired therapeutic effect includes, but is not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis.
  • the CAR immune effector cells of the present invention are used to delay disease development or to slow down the progression of the disease.
  • the term "effective amount” refers to such an amount or dosage of the CAR immune effector cells of the present invention, which is administered to the patient in a single or multiple doses, and produces the desired effect in the patient in need of treatment or prevention.
  • the effective amount can be easily determined by the attending physician as a technician in the field by considering the following factors: such as mammalian species; weight, age and general health; the specific disease involved; the extent or severity of the disease; the response of individual patients; the specific CAR immune effector cells administered; the mode of administration; the bioavailability characteristics of the administered preparation; the selected dosage regimen; and the use of any concomitant therapy.
  • “Therapeutically effective amount” refers to the amount of the desired therapeutic result effectively achieved at the required dosage and for the required time period.
  • the therapeutically effective amount of CAR immune effector cells can be changed according to various factors such as disease state, individual age, sex and weight and the ability of CAR immune effector cells to stimulate the desired response in individuals.
  • the therapeutically effective amount is also such an amount, in which any toxic or harmful effects of CAR immune effector cells are less than the therapeutic beneficial effects.
  • "therapeutically effective amount” preferably suppresses measurable parameters (such as tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60% or 70% and still more preferably at least about 80% or 90%.
  • the ability of CAR immune effector cells to suppress measurable parameters e.g., cancer
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is attached.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which it has been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively attached. Such vectors are referred to herein as "expression vectors.”
  • Subject/patient sample refers to a collection of cells, tissues or body fluids obtained from a patient or subject.
  • the source of the tissue or cell sample can be solid tissue, such as from fresh, frozen and/or preserved organ or tissue samples or biopsy samples or puncture samples; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time of pregnancy or development of the subject.
  • Tissue samples may contain compounds that are naturally not mixed with tissues in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.
  • tumor samples include, but are not limited to, tumor biopsies, fine needle aspirates, bronchial lavage fluid, pleural fluid (pleural effusion), sputum, urine, surgical specimens, circulating tumor cells, serum, plasma, circulating plasma proteins, ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, and preserved tumor samples, such as formalin-fixed, paraffin-embedded tumor samples or frozen tumor samples.
  • IL-13 receptor ⁇ chain isoform 2 (IL13R ⁇ 2) is a receptor polypeptide for IL-13. It is almost exclusively expressed in cancer cells and not in normal tissue cells (except testes). It is highly expressed on the surface of glioma cells and plays an important role in the malignant proliferation of gliomas.
  • IL13R ⁇ 2 IL-13 receptor ⁇ chain isoform 2
  • GBM patients more than 50% of patients express IL13R ⁇ 2, making it an effective target for CAR-T therapy of GBM (Sharma P, Debinski W. Receptor-Targeted Glial Brain Tumor Therapies. International journal of molecular sciences. 2018; 19(11)).
  • the present invention relates to an optimized chimeric antigen receptor (CAR) polypeptide targeting IL13R ⁇ 2.
  • a chimeric antigen receptor (CAR) is a recombinant polypeptide comprising at least an extracellular recognition domain, a transmembrane region, and an intracellular signaling domain.
  • the extracellular recognition domain (also referred to as "extracellular domain") of the CAR polypeptide of the present invention is an optimized anti-IL13R ⁇ 2 scFv sequence that specifically recognizes and binds to IL13R ⁇ 2 on the surface of target cells.
  • CAR can both bind to antigens and transduce T cell activation, and the T cell activation is independent of MHC restriction, CAR can be used to treat antigen-positive tumor patients, regardless of the HLA genotype of the tumor patient.
  • Adoptive immunotherapy of lymphocytes using CAR can be a powerful therapeutic strategy for treating cancer.
  • the optimized chimeric antigen receptor (CAR) polypeptide targeting IL13R ⁇ 2 of the present invention comprises:
  • An optimized anti-IL13R ⁇ 2 scFv sequence wherein the scFv sequence specifically binds to IL13R ⁇ 2 and comprises:
  • a heavy chain complementary determining region CDR H1 represented by the amino acid sequence KYGVH (SEQ ID NO: 15), or a variant of said CDR H1 having no more than 2 amino acid changes or no more than 1 amino acid change;
  • CDR H2 represented by the amino acid sequence VKWAGGSTDTDSALMS (SEQ ID NO:16), or a variant of said CDR H2 having no more than 2 amino acid changes or no more than 1 amino acid change;
  • CDR L light chain complementary determining region 1 represented by the amino acid sequence TASLSVSSTYLH (SEQ ID NO:18), or a variant of said CDR L1 having no more than 2 amino acid changes or no more than 1 amino acid change;
  • CDR L3 represented by the amino acid sequence HQYHRSPLT (SEQ ID NO: 20), or a variant of said CDR L3 having no more than 2 amino acid changes or no more than 1 amino acid change;
  • amino acid change is an addition, deletion or substitution of an amino acid
  • CD8 hinge region (SEQ ID NO 8), or a CD8 hinge region having at least 80% sequence identity thereto.
  • TM transmembrane region
  • a CD28 transmembrane domain or a variant thereof having 1-5 amino acid modifications for example, the sequence shown in SEQ ID NO:9 or a variant thereof having 1-2 amino acid modifications;
  • a CD4 transmembrane domain or a variant thereof having 1-5 amino acid modifications for example, the sequence shown in SEQ ID NO: 10 or a variant thereof having 1-2 amino acid modifications;
  • a CD8 transmembrane domain or a variant thereof having 1-5 amino acid modifications for example, the sequence shown in SEQ ID NO: 11 or a variant thereof having 1-2 amino acid modifications;
  • a CD28 co-stimulatory domain or a variant thereof having 1-5 amino acid modifications for example, the sequence shown in SEQ ID NO: 12 or a variant thereof having 1-2 amino acid modifications;
  • a 4-1BB co-stimulatory domain or a variant thereof having 1-5 amino acid modifications for example, the sequence shown in SEQ ID NO: 13 or a variant thereof having 1-2 amino acid modifications;
  • a stimulatory signaling domain which is a CD3 ⁇ signaling domain or a variant thereof having 1-10 amino acid modifications, for example, a sequence as shown in SEQ ID NO: 14 or a variant thereof having 1-10 or 1-5 amino acid modifications;
  • amino acid modification is the addition, deletion or substitution of amino acids
  • the extracellular domain of the chimeric antigen receptor of the present invention is an optimized anti-IL13R ⁇ 2 scFv sequence that specifically binds to IL13R ⁇ 2, wherein the scFv sequence specifically binds to IL13R ⁇ 2 and comprises:
  • a heavy chain variable region comprising the sequence of SEQ ID NO:2, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and
  • a light chain variable region comprising the sequence of SEQ ID NO:4 or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.
  • the extracellular domain of CAR i.e., the optimized anti-IL13R ⁇ 2 scFv sequence
  • immune effector cells e.g., T cells, NK cells
  • the cells expressing IL13R ⁇ 2 are tumor cells (including glioma cells).
  • the transmembrane domain included in the chimeric antigen receptor of the present invention is an anchored transmembrane domain, which is a component of a polypeptide chain that can be integrated into a cell membrane.
  • the transmembrane domain can be fused with other extracellular and/or intracellular polypeptide domains, whereby these extracellular and/or intracellular polypeptide domains will also be restricted to the cell membrane.
  • the transmembrane domain confers membrane attachment to the CAR polypeptide of the present invention.
  • the CAR polypeptide of the present invention includes at least one transmembrane domain, which may be derived from a natural source or a recombinant source, comprising dominant hydrophobic residues such as leucine and valine.
  • the transmembrane domain may be derived from a membrane-bound protein or a transmembrane protein such as CD4, CD28, CD8 (e.g., CD8 ⁇ , CD8 ⁇ ) transmembrane domain.
  • the transmembrane domain in the chimeric antigen receptor of the present invention is a CD4 transmembrane domain or a variant thereof having 1-10 amino acid modifications, for example, a variant thereof having 1-5 amino acid modifications.
  • the amino acid modification is an addition, deletion or substitution of an amino acid.
  • the CD4 transmembrane domain is the sequence shown in SEQ ID NO: 10.
  • the transmembrane domain in the chimeric antigen receptor of the present invention is a CD8 transmembrane domain or a variant thereof having 1-10 amino acid modifications, for example, a variant thereof having 1-5 amino acid modifications.
  • the amino acid modification is an addition, deletion or substitution of an amino acid.
  • the CD8 transmembrane domain is the sequence shown in SEQ ID NO: 11.
  • the transmembrane domain in the chimeric antigen receptor of the present invention is a CD28 transmembrane domain or a variant thereof having 1-10 amino acid modifications, for example, a variant thereof having 1-5 amino acid modifications.
  • the amino acid modification is an addition, deletion or substitution of an amino acid.
  • the CD28 transmembrane domain is the sequence shown in SEQ ID NO:9.
  • a CAR-T cell comprising a CD28 transmembrane domain that specifically binds to IL13R ⁇ 2 has excellent anti-GBM activity in vivo.
  • the transmembrane domain in the CAR of the present invention is connected to the extracellular region of the CAR (ie, the anti-IL13R ⁇ 2 scFv sequence) by means of a hinge region/spacer.
  • a hinge region/spacer comprises the entire or part of an immunoglobulin (eg, IgG1, IgG2, IgG3, IgG4) hinge region, that is, a sequence falling between the CH1 and CH2 domains of an immunoglobulin, for example, an IgG4Fc hinge or a CD8 hinge.
  • Some hinge regions/spacers comprise an immunoglobulin CH3 domain or both a CH3 domain and a CH2 structure.
  • the sequence derived from an immunoglobulin may comprise one or more amino acid modifications, for example, 1, 2, 3, 4 or 5 substitutions, for example, substitutions that reduce off-target binding.
  • the hinge region/spacer is an IgG hinge region or a variant thereof having 1-2 amino acid modifications, e.g., an IgG4 hinge region or a variant thereof having 1-2 amino acid modifications.
  • the length of the hinge region/spacer region is optimized, and it is found that for IL13R ⁇ 2, a short hinge region/spacer region (for example, the IgG4 hinge region shown in SEQ ID NO:7) is easier to activate CAR-T than a long hinge region/spacer region.
  • the IgG4 hinge region shown in SEQ ID NO:7 does not retain the heavy chain constant region 2 of IgG4 and therefore does not have the ability to bind to Fc receptors (e.g., Fc ⁇ R), thereby avoiding off-target activation of CAR-T cells.
  • Fc receptors e.g., Fc ⁇ R
  • the cytoplasmic domain included in the CAR of the present invention includes an intracellular signaling domain.
  • the intracellular signaling domain can activate at least one effector function of the immune cell introduced with the CAR of the present invention.
  • intracellular signaling domains used in the CAR of the present invention include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that act synergistically to initiate signal transduction after the extracellular domain binds to IL13R ⁇ 2, as well as any derivatives or variants of these sequences and any recombinant sequences with the same functional capacity.
  • TCRs T cell receptors
  • co-receptors that act synergistically to initiate signal transduction after the extracellular domain binds to IL13R ⁇ 2, as well as any derivatives or variants of these sequences and any recombinant sequences with the same functional capacity.
  • the CAR of the present invention is also designed to generate a costimulatory signal domain (CSD).
  • CSD costimulatory signal domain
  • the activation of T cells is mediated by two different cytoplasmic signaling sequences: those sequences (primary intracellular signaling domains) that initiate antigen-dependent primary activation through TCR and those sequences (secondary cytoplasmic domains, e.g., costimulatory domains) that act in an antigen-independent manner to provide costimulatory signals.
  • the CAR of the present invention comprises a primary intracellular signaling domain, for example, a primary signaling domain of CD3 ⁇ , for example, a CD3 ⁇ signaling domain shown in SEQ ID NO: 14 or a variant thereof having 1-10 amino acid modifications, for example, a variant thereof having 1-5 amino acid modifications, wherein the amino acid modification is an addition, deletion or substitution of an amino acid.
  • the intracellular signaling domain in the CAR of the present invention also includes a secondary signaling domain (i.e., a co-stimulatory signaling domain).
  • the co-stimulatory signaling domain refers to the CAR portion that includes the intracellular domain of a co-stimulatory molecule.
  • Co-stimulatory molecules are cell surface molecules required for immune effector cells to effectively respond to antigens in addition to antigen receptors or their ligands.
  • co-stimulatory molecules include but are not limited to CD28, 4-1BB (CD137), and OX40, the co-stimulatory effects of which enhance the proliferation, effector function, and survival of human CAR T cells in vitro and enhance the anti-tumor activity of human T cells in vivo.
  • the costimulatory signaling domain in the chimeric antigen receptor of the present invention is a CD28 costimulatory domain or a variant thereof having 1-10 amino acid modifications, for example, a variant thereof having 1-5 amino acid modifications.
  • the amino acid modification is the addition, deletion or substitution of an amino acid.
  • the costimulatory signaling domain in the chimeric antigen receptor of the present invention is a 4-1BB costimulatory domain or a variant thereof with 1-10 amino acid modifications, for example, a variant with 1-5 amino acid modifications.
  • the amino acid modification is the addition, deletion or substitution of an amino acid.
  • the intracellular region of the CAR of the present invention comprises multiple co-stimulatory domains in series with CD3 ⁇ , such as a CD28 co-stimulatory domain and a 4-1BB co-stimulatory domain, and when the CAR is expressed on the surface of an immune effector cell (e.g., a T cell, a NK cell), the CAR enables the T cell to receive a co-stimulatory signal.
  • an immune effector cell e.g., a T cell, a NK cell
  • Intracellular signaling sequences of the CAR of the present invention can be connected to each other in a random order or in a specified order.
  • short oligopeptide linkers or polypeptide linkers can form bonds between intracellular signaling sequences.
  • a glycine-serine doublet can be used as a suitable linker.
  • a single amino acid, for example, alanine, glycine can be used as a suitable linker.
  • the intracellular signaling domain of the CAR of the present invention is designed to include the costimulatory signaling domain of CD28, the costimulatory signaling domain of 4-1BB, and the stimulatory signaling domain of CD3 ⁇ .
  • the CAR polypeptides of the present invention can also be modified so that the amino acid sequence is changed, but the desired activity is not changed.
  • the CAR polypeptide can be subjected to additional amino acid substitutions that result in amino acid substitutions at "non-essential" amino acid residues.
  • a non-essential amino acid residue in a molecule can be replaced with another amino acid residue from the same side chain family.
  • an amino acid fragment can be replaced with a structurally similar fragment that differs in the order and composition of the side chain family members, for example, a conservative substitution can be performed in which an amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid
  • the present invention also contemplates the generation of functionally equivalent CAR polypeptide molecules.
  • the present invention provides nucleic acid molecules encoding CAR constructs described herein.
  • the nucleic acid molecule is provided as a DNA construct.
  • the construct encoding the CAR of the present invention can be obtained using recombinant methods known in the art.
  • the target nucleic acid can be produced synthetically rather than by genetic recombination methods.
  • the present invention also provides a vector inserted with a CAR construct of the present invention.
  • a vector By effectively connecting a nucleic acid encoding a CAR polypeptide to a promoter and incorporating the construct into an expression vector, expression of a natural or synthetic nucleic acid encoding CAR is achieved.
  • the vector may be suitable for replication and integration in eukaryotic organisms. Common cloning vectors contain transcription and translation terminators, initiation sequences, and promoters for regulating the expression of the desired nucleic acid sequence.
  • retroviruses provide a convenient platform for gene delivery systems.
  • the CAR construct of the present invention can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to the subject's cells in vivo or in vitro.
  • Numerous retroviral systems are known in the art.
  • a lentiviral vector is used.
  • the nucleic acid sequence of the CAR construct of the present invention is cloned into a lentiviral vector to produce a full-length CAR construct in a single coding frame, and the EF1 ⁇ promoter is used for expression.
  • Retroviral vectors can also be, for example, ⁇ retroviral vectors.
  • ⁇ retroviral vectors can, for example, include promoters, packaging signals ( ⁇ ), primer binding sites (PBS), one or more (e.g., two) long terminal repeats (LTR) and target transgenics, for example, genes encoding CAR.
  • ⁇ retroviral vectors can lack viral structural genes such as gag, pol and env.
  • a promoter capable of expressing a CAR transgene in mammalian T cells is the EF1a promoter.
  • the natural EF1a promoter drives the expression of the ⁇ subunit of the elongation factor-1 complex, which is responsible for enzymatic delivery of aminoacyl tRNA to the ribosome.
  • the EF1a promoter has been widely used in mammalian expression plasmids and has been shown to effectively drive the expression of CAR from a transgene cloned into a lentiviral vector. See, for example, Milone et al., Mol. Ther. 17 (8): 1453–1464 (2009).
  • CMV immediate early cytomegalovirus
  • This promoter sequence is a constitutive strong promoter sequence that can drive any polynucleotide sequence effectively connected thereto to express at a high level.
  • other constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter and human gene promoter, such as but not limited to actin promoter, myosin promoter, elongation factor-1 ⁇ promoter, hemoglobin promoter and creatine kinase promoter.
  • the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention.
  • the present invention provides methods for expressing a CAR construct of the present invention in a mammalian immune effector cell (e.g., a mammalian T cell or a mammalian NK cell) and the immune effector cells produced thereby.
  • a mammalian immune effector cell e.g., a mammalian T cell or a mammalian NK cell
  • a cell source e.g., immune effector cells, e.g., T cells or NK cells
  • T cells can be obtained from numerous sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors.
  • T cells from blood components collected from a subject can be obtained by apheresis.
  • Apheresis products generally contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes and platelets.
  • the cells collected by apheresis can be washed to remove the plasma fraction and to place cells in a suitable buffer or culture medium for subsequent processing steps.
  • cells are washed with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • Specific T cell subsets such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques.
  • the time period is about 30 minutes to 36 hours or longer.
  • Longer incubation time can be used to separate T cells in any case where there is a small amount of T cells, such as for separating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immunocompromised individuals.
  • TIL tumor infiltrating lymphocytes
  • the efficiency of capturing CD8+T cells can be increased using longer incubation time.
  • T cell subsets can be preferentially selected at the beginning of culture or at other time points during the culture process.
  • Enrichment of T cell populations can be accomplished by a negative selection process using a combination of antibodies directed against surface markers unique to the negatively selected cells.
  • One approach is to sort and/or select cells using negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed against cell surface markers present on the negatively selected cells.
  • the immune effector cell can be an allogeneic immune effector cell, e.g., a T cell or a NK cell.
  • the cell can be an allogeneic T cell, e.g., an allogeneic T cell lacking expression of a functional T cell receptor (TCR) and/or human leukocyte antigen (HLA) (e.g., HLA class I and/or HLA class II).
  • TCR functional T cell receptor
  • HLA human leukocyte antigen
  • a T cell lacking a functional TCR can, for example, be engineered so that it does not express any functional TCR on its surface; engineered so that it does not express one or more subunits that make up a functional TCR (e.g., engineered so that it does not express or shows reduced expression of TCR ⁇ , TCR ⁇ , TCR ⁇ , TCR ⁇ , TCR ⁇ and/or TCR ⁇ ); or engineered so that it produces very few functional TCRs on its surface.
  • the T cells described herein can be engineered, for example, so that it does not express functional HLA on its surface.
  • the T cells described herein can be engineered so that cell surface expression of HLA (e.g., HLA class I and/or HLA class II) is downregulated.
  • HLA e.g., HLA class I and/or HLA class II
  • downregulation of HLA can be achieved by reducing or eliminating beta-2 microglobulin (B2M) expression.
  • the T cell may lack a functional TCR and a functional HLA, e.g., HLA class I and/or HLA class II.
  • cells transduced with a nucleic acid encoding a CAR described herein are proliferated, for example, the cells are proliferated in culture for 2 hours to about 14 days.
  • the CAR-expressing immune effector cells obtained after in vitro proliferation can be tested for effector function as described in the examples.
  • the CAR-T cells of the present invention can maintain the killing activity against tumors expressing IL13R ⁇ 2.
  • the optimized third-generation chimeric antigen receptor (CAR) polypeptide targeting IL13R ⁇ 2 of the present invention has excellent safety and in vivo anti-GBM activity due to its high selectivity and specific targeting of IL13R ⁇ 2.
  • the present invention obtains immune effector cells expressing the CAR polypeptide of the present invention by optimizing the structure of the CAR construct, which is used to treat solid tumors associated with the expression of IL13R ⁇ 2, such as gliomas, in subjects.
  • engineered T cells e.g., patient-specific autologous T cells
  • ACT adoptive cell therapy
  • a variety of T cell subsets can be used to express the CAR polypeptides of the present invention.
  • the immune effector cells when using immune effector cells expressing the CAR polypeptides of the present invention to treat patients, can be autologous T cells or allogeneic T cells.
  • the T cells used are CD4+ and CD8+ central memory T cells (T CM ), which are CD45RO+CD62L+, and the use of such cells can improve the long-term survival of cells after adoptive transfer compared to the use of other types of patient-specific T cells.
  • NK cells are engineered to express the CAR polypeptides of the present invention. After amplifying the other immune cells (e.g., NK cells) engineered, they are used for adoptive cell therapy (ACT).
  • ACT adoptive cell therapy
  • the immune effector cells expressing the CAR polypeptide of the present invention are used to treat cancer that expresses or overexpresses IL13R ⁇ 2 in a subject, and are capable of reducing the severity of at least one symptom or indication of cancer or inhibiting cancer cell growth.
  • the present invention provides a method for treating a disease associated with the expression of IL13R ⁇ 2 (e.g., a cancer that expresses or overexpresses IL13R ⁇ 2) in a subject, comprising administering to an individual in need thereof a therapeutically effective amount of immune effector cells expressing a CAR polypeptide of the present invention.
  • a disease associated with the expression of IL13R ⁇ 2 e.g., a cancer that expresses or overexpresses IL13R ⁇ 2
  • administering to an individual in need thereof a therapeutically effective amount of immune effector cells expressing a CAR polypeptide of the present invention.
  • the present invention provides the use of the aforementioned immune effector cells expressing the CAR polypeptide of the present invention in the preparation of a medicament for treating a disease associated with the expression of IL13R ⁇ 2 (eg, a cancer expressing or overexpressing IL13R ⁇ 2).
  • a disease associated with the expression of IL13R ⁇ 2 eg, a cancer expressing or overexpressing IL13R ⁇ 2.
  • Immune effector cells expressing a CAR polypeptide of the invention can also be administered to individuals whose cancer has been treated with one or more prior therapies but has subsequently relapsed or metastasized.
  • immune effector cells e.g., T cells, NK cells
  • a CAR polypeptide of the invention are used for parenteral, transdermal, intracavitary, intraarterial, intravenous, intrathecal administration, or directly injected into a tissue or tumor.
  • the immune effector cells expressing the CAR polypeptides of the present invention can be administered to the subject at a suitable dose.
  • the dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical field, the dosage for any one patient depends on many factors, including the patient's weight, body surface area, age, specific compound to be administered, sex, administration time and route, general health status, and other drugs to be administered in parallel.
  • immune effector cells e.g., T cells, NK cells
  • a CAR polypeptide of the invention are administered parenterally, preferably intravenously, in a single or multiple doses of 1 ⁇ 10 6 to 1 ⁇ 10 12 immune effector cells, preferably 1 ⁇ 10 7 to 1 ⁇ 10 10 immune effector cells, for example 5 ⁇ 10 7 , 1 ⁇ 10 8 , 5 ⁇ 10 8 , 1 ⁇ 10 9 , 5 ⁇ 10 9 immune effector cells.
  • immune effector cells expressing the CAR polypeptides of the present invention are administered to individuals with cancer, resulting in the complete disappearance of the tumor. In some embodiments, immune effector cells expressing the CAR polypeptides of the present invention are administered to individuals with cancer, resulting in a reduction of tumor cells or tumor size by at least 85% or more.
  • the reduction of tumors can be measured by any method known in the art, such as X-rays, positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), cytology, histology, or molecular genetic analysis.
  • GBM cell lines U251, U373, U87 and retroviral packaging cell lines PG13 and Phoenix ECO were purchased from the American Type Culture Collection (ATCC).
  • U251 cells and U373 cells were transduced with retrovirus to express the green fluorescent protein (GFP) gene and the firefly luciferase (LUC) reporter gene (sometimes abbreviated as "GL”).
  • GBM cell lines grow well in DMEM medium (Lonza) containing 10% fetal bovine serum (FBS, Biosera), 100 U/mL penicillin, and 100 ⁇ g/mL streptomycin (EallBio Life Sciences).
  • Retroviral production cell lines were cultured in DMEM medium containing 10% FBS without penicillin and streptomycin.
  • Flow cytometry was performed using a BD FacsCanto II Plus instrument (BD Biosciences), and the results were analyzed using FlowJo v.10 software (Tree star, Inc. Ashland, OR).
  • the antibodies used were as follows: anti-human CD3-APC-R700 antibody (BD Bioscience), anti-human-CD4-V450 (BD Bioscience), anti-human-CD8-PE-Cy7 (BD Bioscience), anti-human IL13Ra-APC (BD Bioscience), and goat anti-mouse IgG-APC (Sigma).
  • CAR-T cells and target cells were co-cultured at an E:T ratio of 10:1 for 24 h, and the killing of target cells was detected by flow cytometry.
  • CAR-T cells were co-cultured with human GBM cell lines (U373, U251, and U87) at an E:T ratio of 10:1 for 24 hours.
  • the expression of human interferon- ⁇ (IFN- ⁇ ), tumor necrosis factor- ⁇ (TNF- ⁇ ), IL17A, IL4, IL6, and IL10 in the supernatant of co-cultured cells was detected using a commercial flow cytometry bead array (CBA) kit (BD Biosciences). The specific operation steps were performed according to the instructions of the kit.
  • CBA flow cytometry bead array
  • RTCA Real-time cell analysis
  • the proliferation/cytotoxicity of CAR T cells was evaluated using the xCELLigence RTCA system (Roche Applied Science, Basel, Switzerland). The system is based on a gold plate sensor electrode with electrical impedance reading, located at the bottom of the cytotoxicity plate (E-16 plate).
  • E-16 plate a gold plate sensor electrode with electrical impedance reading, located at the bottom of the cytotoxicity plate (E-16 plate).
  • target cells U251-GL or target cells U373-GL were seeded in E-16 plates at 1 ⁇ 104 cells per well.
  • 1 ⁇ 105 CAR-T cells were added to the E-16 plate and incubated with human GBM cells and monitored every 15 min to obtain the cell index for 48 h. Each independent experiment was performed in triplicate. The interval slope was automatically calculated using the RTCA software to evaluate the rate of change of the cell index.
  • the cell index was normalized to equal values at standardized time points.
  • mice used in the experiment were 6-8 week old NOD-SCID mice purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. 2 ⁇ 10 5 U251-GL cells and U373-GL cells were injected into the right striatum of female NOD-SCID mice to construct an orthotopic xenograft mouse model, which was recorded as day 1. On day 6, 3 ⁇ 10 7 CAR-T cells were injected through the tail vein. The development of the tumor was monitored using a small animal imaging instrument (IVIS, Xenogen, Alameda, CA, USA), and the mice were killed when the tumor diameter reached 20 mm. The animal experiment was approved by the Ethics Committee of Beijing Century Altar Hospital.
  • IVIS small animal imaging instrument
  • the CAR targeting IL13R ⁇ 2 shown in FIG. 1 and comprising an optimized scFv sequence for IL13R ⁇ 2 was constructed using a retroviral vector.
  • the CAR targeting IL13R ⁇ 2 contains an optimized anti-IL13R ⁇ 2 scFv sequence located in the extracellular domain, an optimized IgG4 hinge region, a CD28 transmembrane region, a CD28 co-stimulatory domain located in the intracellular part, a 4-1BB co-stimulatory domain, and a CD3 ⁇ signaling domain.
  • Thermo Company's GeneArt gene synthesis technology was used to synthesize a nucleotide sequence encoding the anti-IL13R ⁇ 2 scFv sequence, which, from N-terminus to C-terminus, consists of the antibody heavy chain variable region VH nucleotide sequence shown in SEQ ID NO:1, the nucleotide sequence encoding the linker shown in SEQ ID NO:5, and the antibody light chain variable region VL nucleotide sequence shown in SEQ ID NO:3.
  • nucleotide sequences encoding the IgG4 hinge region shown in SEQ ID NO:7, the CD28 transmembrane domain shown in SEQ ID NO:9, the CD28 co-stimulatory domain shown in SEQ ID NO:12, the 4-1BB co-stimulatory domain shown in SEQ ID NO:13, and the CD3 ⁇ signaling domain shown in SEQ ID NO:14 were ligated into the retroviral vector SFG (Addgene, also known as "vector pMSGV1" to obtain the vector retro-SFG-IgG4.
  • the constructed IL13R ⁇ 2-specific CAR retroviral vector was used for virus packaging. Specifically, the retroviral vector retro-SFG-IL13R ⁇ 2 scFv-IgG4-CD28-4-1BB-CD3 ⁇ was co-transfected into PG13 cells using a calcium phosphate reagent. The culture medium was replaced 48 hours after transfection, and the culture supernatant was collected. The culture supernatant was collected again 72 hours later and filtered through 0.45 ⁇ m. Syringe filter to obtain the retroviral stock solution.
  • the virus stock solution was ultracentrifuged at 4°C and 32,000 r/min for 2 hours, and the retrovirus precipitate was dissolved in X-VIVO culture medium to obtain the retrovirus concentrate, which was aliquoted and stored at -80°C.
  • the titer of the retroviral concentrate was tested. 0.5 ⁇ 10 6 Jurkat cells were plated in each well of a 96-well pointed bottom plate, 200 ⁇ L/well, the retroviral concentrate was diluted 100 times, and 400 ⁇ L, 40 ⁇ L and 10 ⁇ L were added to each well at a ratio of 1:50, 1:500 and 1:2000. Centrifuge at 32°C and 1200 ⁇ g for 90 min. After 4 hours, the cells were washed once with DPBS and plated on a 12-well plate, and detected by flow cytometry 48 hours after transduction. The results showed that the retroviral concentrate prepared in this example was able to infect Jurkat cells and express CAR after being diluted 100 times. Therefore, the production of CAR-T cells was implemented using the retroviral concentrate.
  • Example 1 the concentrated retrovirus solution harvested in Example 1 was used to transduce T cells, thereby preparing CAR-T cells.
  • PBMC mononuclear cells
  • GE-healthcare lymphocyte separation fluid
  • T cells in PBMC were stimulated with anti-CD3/CD28 T cell activator Dynabeads (Invitrogen). After 48 hours of stimulation, T cells were transfected with the retroviral concentrate of Example 1.
  • the retroviral transfection experiment was performed with reference to the instructions of the calcium phosphate transfection kit (Sigma). On day 7, flow cytometry was used to detect the positive rate of CAR expression in transfected T cells.
  • CAR-T cells were cultured in X-VIVO-15 medium containing 5% human AB serum (SIGMA), 100U/ml IL-2, 100U/ml penicillin, and 100 ⁇ g/ml streptomycin (EallBio Life Sciences). This study was approved by the Institutional Review Board of Beijing Century Altar Hospital, and informed consent was obtained from all participants.
  • SIGMA human AB serum
  • 100U/ml IL-2 100U/ml
  • penicillin 100U/ml
  • streptomycin EallBio Life Sciences
  • T cells from healthy donors and activated by CD3/CD28 antibodies were transduced with the retrovirus prepared in Example 1, and the gene-modified T cells were detected by FACS analysis 7 days later.
  • the CAR containing the anti-IL13R ⁇ 2 scFv sequence was stably expressed on the surface of T cells.
  • the killing effect of CAR-T cells on tumor cells can be detected by the degranulation ability of CAR-T cells, that is, the expression of CD107a.
  • the CAR-T cells containing the anti-IL13R ⁇ 2 scFv sequence produced in Example 2 contain high concentrations of cytotoxic granules in the form of vesicles, such as perforin, granzyme, etc.
  • Lysosomal associated membrane protein-1 (LAMP-1) also known as CD107a, is a marker of cell degranulation. Under the stimulation of tumor cells, the expression of CD107a is associated with the lysis of target cells mediated by the CAR-T cells produced in Example 2.
  • the CD107a expression rate was positively correlated with the killing activity of the CAR-T cells.
  • Example 5 Determination of cytokines produced by CAR-T cells containing anti-IL13R ⁇ 2 scFv sequences
  • CAR-T cells containing anti-IL13R ⁇ 2 scFv sequence and IL13R ⁇ 2-positive human GBM cell lines (U373, U251, and U87 cells) were co-cultured at an E:T ratio of 10:1 for 24 hours, interferon- ⁇ (IFN- ⁇ ) and tumor necrosis factor- ⁇ (TNF- ⁇ ) in the culture supernatant were detected by CBA method, wherein IFN- ⁇ and TNF- ⁇ were positively correlated with the killing ability of CAR-T cells on target cells.
  • IFN- ⁇ interferon- ⁇
  • TNF- ⁇ tumor necrosis factor- ⁇
  • the cell growth analysis of tumor cells was performed using a real-time cell analyzer (RTCA).
  • RTCA real-time cell analyzer
  • a micro-gold electronic sensor chip is integrated at the bottom of the detection plate E-Plate 16.
  • Figures 10 and 11 show the cell growth results of tumor cells detected by the RTCA method, and the anti-tumor ability of CAR-T cells containing anti-IL13R ⁇ 2 scFv sequences was evaluated in vitro.
  • the number of tumor cells in the U373 tumor cell control group increased significantly with the extension of time; the number of tumor cells in the co-culture group of CAR-T cells containing anti-IL13R ⁇ 2 scFv sequence and U373 tumor cells ( ⁇ line) decreased significantly after 20 hours of co-culture, and the CAR-T cells significantly killed the tumor cells.
  • the number of tumor cells in the U251 tumor cell control group increased significantly with the extension of time; after 20 hours of co-culture with U251 tumor cells in the CAR-T cells containing the anti-IL13R ⁇ 2 scFv sequence ( ⁇ line), the number of tumor cells decreased significantly, and the CAR-T cells had a significant killing effect on tumor cells.
  • the CAR-T cells of the present invention containing the anti-IL13R ⁇ 2 scFv sequence significantly inhibited the growth of U373 and U251 tumor cells and had obvious anti-tumor activity.
  • an orthotopic xenograft mouse model was established by intracranial injection of glioma cells.
  • Glioma cell lines U373 and U251 express IL13R ⁇ 2 on their cell surface.
  • An orthotopic xenograft mouse model was constructed using glioma cell lines U373 and U251 cells. Specifically, the mice used in the experiment were six to eight-week-old NOD-SCID mice, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. 2 ⁇ 10 5 U251-GL cells or U373-GL cells were injected into the right striatum of female NOD-SCID mice (recorded as the first day, also abbreviated as D1).
  • a positive control group was set up in the experiment, using IL13-CAR-T.
  • the preparation of the positive control IL13-CAR-T cells was similar to that of Example 1 and Example 2, except that the anti-IL13R ⁇ 2 scFv sequence was replaced by the IL13 sequence shown in SEQ ID NO:24.
  • Tumor growth was observed by detecting fluorescence signals using a small animal imaging instrument (IVIS, Xenogen, Alameda, CA, USA) to monitor tumor development, and mice were killed when the tumor diameter reached 20 mm. Animal experiments were approved by the Ethics Committee of Beijing Shijitan Hospital.
  • IVIS small animal imaging instrument
  • the longest survival period of the U251 in situ animal model treated with IL13-CAR-T in the positive control group was 34 days, while the longest survival period of mice treated with IL13R ⁇ 2-CAR-T of the present invention was 50 days.
  • the longest survival period of the U373 in situ animal model treated with IL13-CAR-T in the positive control group was 60 days, while the longest survival period of mice treated with IL13R ⁇ 2-CAR-T of the present invention was 64 days.

Abstract

L'invention concerne un récepteur antigénique chimérique ciblant IL13Rα2, comprenant : (1) une séquence scFv anti-IL13Rα2 optimisée; (2) une région charnière/région d'espaceur optimisée; (3) une région transmembranaire (TM); (4) un domaine costimulateur CD28 et un domaine costimulateur 4-1BB; et (5) un domaine de signalisation CD3ζ. L'invention concerne également une molécule d'acide nucléique codant pour le récepteur antigénique chimérique, un vecteur comprenant la molécule d'acide nucléique, une cellule effectrice immunitaire (par exemple, une cellule T ou une cellule NK) modifiée pour exprimer le récepteur antigénique chimérique, et une utilisation de la cellule effectrice immunitaire modifiée dans le traitement de maladies associées à l'expression d'IL13Rα2, par exemple, un gliome.
PCT/CN2022/136205 2022-09-29 2022-12-02 RÉCEPTEUR ANTIGÉNIQUE CHIMÉRIQUE OPTIMISÉ CIBLANT IL13Rα2 ET SON UTILISATION WO2024066026A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104936621A (zh) * 2012-11-07 2015-09-23 辉瑞公司 抗IL-13受体α2抗体和抗体-药物缀合物
CN107002084A (zh) * 2014-09-19 2017-08-01 希望之城公司 靶向IL13Rα2的共刺激嵌合抗原受体T细胞
CN112236151A (zh) * 2018-03-14 2021-01-15 西雅图儿童医院（Dba西雅图儿童研究所） 用于肿瘤特异性T细胞免疫疗法的IL-13受体α2(IL13RA2)嵌合抗原受体
WO2021154543A2 (fr) * 2020-01-31 2021-08-05 City Of Hope LYMPHOCYTES T MODIFIÉS PAR UN RÉCEPTEUR CHIMÈRE D'ANTIGÈNE CIBLÉ POUR LE TRAITEMENT DES MALIGNITÉS POSITIVES À IL13Rα2
CN114014941A (zh) * 2022-01-10 2022-02-08 卡瑞济(北京)生命科技有限公司 靶向IL13Rα2的嵌合抗原受体及其用途

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104936621A (zh) * 2012-11-07 2015-09-23 辉瑞公司 抗IL-13受体α2抗体和抗体-药物缀合物
CN107002084A (zh) * 2014-09-19 2017-08-01 希望之城公司 靶向IL13Rα2的共刺激嵌合抗原受体T细胞
CN112236151A (zh) * 2018-03-14 2021-01-15 西雅图儿童医院（Dba西雅图儿童研究所） 用于肿瘤特异性T细胞免疫疗法的IL-13受体α2(IL13RA2)嵌合抗原受体
WO2021154543A2 (fr) * 2020-01-31 2021-08-05 City Of Hope LYMPHOCYTES T MODIFIÉS PAR UN RÉCEPTEUR CHIMÈRE D'ANTIGÈNE CIBLÉ POUR LE TRAITEMENT DES MALIGNITÉS POSITIVES À IL13Rα2
CN114014941A (zh) * 2022-01-10 2022-02-08 卡瑞济(北京)生命科技有限公司 靶向IL13Rα2的嵌合抗原受体及其用途

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